Glycosyltransferases from oral bacteria play a fundamental role in dental caries formation by catalyzing the glycosyltransfer of a glucosyl or fructosyl moiety from sucrose to polysaccharide chains. The polysaccharides provide a scaffolding in dental plaque which aids bacterial colonization of smooth enamel surfaces. We have purified one glycosyltransferase (alpha-1,6 glucan: D-fructose-2-glucosyltransferase) referred to by the trivial name, dextransucrase. The enzyme has high specific activity, synthesizes water soluble but not water insoluble dextran, has no levansucrase, sucrose phosphorylase, invertase, or dextranase activity. Detailed kinetic characterization of the enzyme shows that in addition to dextran synthesis, the enzyme catalyzes fructose isotope exchange and sucrose hydrolysis. The enzyme appears to have an absolute requirement for dextran. In the absence of dextran, only sucrose hydrolysis occurs. However, the rate of sucrose hydrolysis is inversely dependent on the dextran concentration so that at high dextran concentrations, where dextran synthesis rates are maximal, sucrose hydrolysis rates approach zero. Steady-state kinetics, product inhibition and isotope exchange kinetics are consistent with a hybrid rapid equilibrium random/ping-pong mechanism. The mechanism implies the participation of a covalent glucosyl-enzyme intermediate. The intermediate has been isolated in preliminary studies. Proposed studies include further characterization of the enzyme intermediate by measuring the rates of release of the glucosyl moiety under alkaline conditions, identification of the anomeric form of the glucosidic bond and isolation of the active site peptide. Additional proposed studies include detailed analysis of pH and temperature effects on catalysis aimed at identification of ionizable groups on the enzyme involved in catalysis; and synthesis of 19 sucrose and fructose analogs to help establish the steric requirements of the active site.